|Publication number||US6022500 A|
|Application number||US 08/756,879|
|Publication date||Feb 8, 2000|
|Filing date||Sep 25, 1996|
|Priority date||Sep 27, 1995|
|Publication number||08756879, 756879, US 6022500 A, US 6022500A, US-A-6022500, US6022500 A, US6022500A|
|Inventors||Vijay T John, Gary L McPherson, Joseph A Akkara, David L Kaplan|
|Original Assignee||The United States Of America As Represented By The Secretary Of The Army|
|Export Citation||BiBTeX, EndNote, RefMan|
|Patent Citations (2), Non-Patent Citations (6), Referenced by (68), Classifications (14), Legal Events (6)|
|External Links: USPTO, USPTO Assignment, Espacenet|
The invention described herein may be manufactured, used and licensed by or for the U.S. Government for U.S. Governmental purposes without the payment to us of any royalty thereon. This invention was made with governmental support from the U.S. Government, U.S. Army Soldier Systems Command, Natick, Mass. under Contract No. DAAK60-93-C-0005.
This application claims benefit from U.S. Provisional Application Ser. No. 60/004,377 filed Sept. 27, 1995.
The present invention relates to polymer microspheres suitable for the encapsulation, stabilization and release of biologically active molecules such as enzymes, drugs, biocides and the like. The present invention also relates to advanced materials, especially composites of polymers with organic or inorganic compounds.
In the drawings:
FIG. 1 is a simplified schematic of enzymatic polymerization using p-ethylphenol;
FIG. 2(a) is a representation of the structural formula of bis(2-ethylhexyl) sodium sulfosuccinate, a surfactant commonly known as AOT;
FIG. 2(b) is a diagram of a reversed micelle depicting the relationship of monomer, surfactant, microaqueous phase and organic solvent bulk phase;
FIG. 3 is a diagram of the reversed micelle reaction system before and after the polymerization reaction illustrating enzyme entrapment;
FIG. 4 is a scanning electron micrograph (SEM) of the polymer microsphere particles formed the polymerization reaction;
FIG. 5 is a phase diagram of AOT and p-ethylphenol with open circle indicating the compositions suitable for formation of polymer microspheres;
FIG. 6 is a transmission electron micrograph (TEM) of polymer particles formed after a five minutes polymerization reaction in which 25% of the monomer was converted;
FIG. 7 is a transmission electron micrograph (TEM) of polymer particles showing gold-labeled peroxidase in the polymer matrix;
FIG. 8(a) is a transmission electron micrograph (TEM) of polymer particles formed after a thirty minutes polymerization reaction;
FIG. 8(b) is a transmission electron micrograph (TEM) of polymer particles formed after a polymerization reaction sufficient to achieve full denisification;
FIG. 9(a) is a transmission electron micrograph (TEM) of polymer particles with a hollow interior formed by sonication of the polymer microspheres;
FIG. 9(b) is another transmission electron micrograph (TEM) of polymer particles with a hollow interior formed by sonication of the polymer microspheres;
FIG. 10 is a transmission electron micrograph (TEM) of a cross-section of a polymer microsphere containing ferrite nanoparticles;
FIG. 11 (a) is a graphical representation of magnetization vs. field loops of a polymer-ferrite composite at a temperature of 100 degrees Kelvin;
FIG. 11 (b) is a graphical representation of magnetization vs. field loops of a polymer-ferrite composite at a temperature of 4.5 degrees Kelvin;
FIG. 12 is representation of the structural formulas of other monomers suitable for enzymatic polymerization;
FIG. 13 is a graphical representation of the fluorescence spectra of 2-naphthol and 1-hydroxypyrene polymers and copolymers, excitation at 413 nm;
FIG. 14 is a graphical representation of the absorbance spectra of cytochrome c, showing polymerization and encapsulation of the enzyme by the reduction in the absorbance peak at 420 nm as the microspheres precipitate out of the micellar supernatant; and
FIG. 15 is a graphical representation of the activity of phosphodiesterase (measured by the hydrolysis of (bis) p-nitrophenylphosphate to form p-nitrophenol as monitored by absorbance at 405 nm) as free enzyme (circles) and encapsulated in polymer microspheres (squares).
We have discovered a novel method of encapsulating enzymes, other solutes or nanoparticles in a polymer matrix. A suitable polymer matrix is comprised of phenolic polymers. The phenolic polymers may be synthesized by conventional chemical processes or by enzyme catalyzed synthesis. The present invention provides composite materials that have numerous applications, including the controlled release of drugs, formulation of protective coatings and paint, and fabricating advanced materials such as nonlinear optical materials.
Polymers based on phenols and aromatic amines can be synthesized enzymatically. The polymers are enzymatically synthesized in reversed micelles. Reversed micelles are microdroplets of water sustained in an oil phase by surfactants to form a water-in-oil emulsion. Reversed micelles are suitable for solubilizing enzymes that require an aqueous environment for optimal catalytic activity. The phenolic monomers used in the present invention are amphiphilic, and partition to the oil-water interface resulting in a organized alignment of the monomers prior to polymerization. Examples of such phenolic monomers are p-ethylphenol, naphthols, and hydroxypyrenes.
The enzyme peroxidase catalyzes the oxidative coupling of a variety of substrates including phenols through activation by hydrogen peroxide. A peroxidase is thus suitable to catalyze the polymerization of polymers such as phenolic polymers. Horseradish peroxidase (HRP) is a suitable peroxidase for the polymerization of phenols and of aromatic amines.
The reversed micelle forms an environment that serves as a template during polymerization. It is believed that interactions with the surfactant at the oil-water interface encourage the folding of the polymer chains to form a mesh that conforms to the shape of the reversed micelle. As the process proceeds, the reversed micelles become joined to form larger structures called microspheres.
Materials formed by the method of the present invention display novel and unexpected properties. Enzymes thus trapped in the polymer matrix, are catalytically active. Nanoparticles entrapped in the matrix confer novel properties to the composite, for example, superparamagnetism.
A number of applications are described herein. The first application is stabilization and maintenance of enzyme activity by encapsulation in the polymer matrix. The second application is the encapsulation of nanoparticles in the matrix, with applications to advanced nanocomposite materials. The third application is the formulation of hollow microspheres for controlled release of compounds. The fourth application is the preparation of highly luminescent (both photoluminescent and electroluminescent polymers.
Phenolic polymers have a variety of conventional applications as resins for coatings, laminates, and the like. The traditional technology for making these polymers involves a formaldehyde-based high temperature process, where undesirable side reactions lead to poor control of polymer structure and molecular weight. In addition, concern over the toxicity of formaldehyde has led to the search for alternative technologies to produce such polymers.
An example is their relevance to nonlinear optics (NLO). In NLO materials, higher order terms become significant in the expansion of the material polarization in terms of an applied electric field. While details of the physics behind NLO materials can be found in several excellent sources (e.g. Prasad and Williams), the relevance of NLO polymers to applications in optical switching, waveguide technology, laser protection, etc. is well-recognized.
When the reaction is conducted in aqueous media, the limited solubility of the monomer and the even lower solubility of the dimer and trimer limit the synthesis of polymers of useful molecular weight. Hence, it is important to conduct the reaction in organic solvents that dissolve oligomers as well as the monomer. Much work has therefore been devoted to peroxidase activity in monophasic organic solvents.
The present invention provides a method for the encapsulation of solutes or particles by the formation of a polymer mesh in reversed micelles.
FIG. 1 is a simplified schematic of the reaction mechanism illustrating the formation of a phenoxy radical which migrates to the ortho position followed by orthocoupling. The rate of H2 O2 addition and the way it is dispersed throughout the system becomes crucial to enzyme viability.
FIG. 1 also illustrates the relevance of the polymer to electrooptics. In contrast to traditional phenol-formaldehyde polymers, where there is a methylene group between the rings, the rings are directly connected in the enzymatically-synthesized material. The polymer is thus conjugated and the resulting electron delocalization along the polymer backbone confers electrooptical properties to the material.
FIGS. 2 and 3 illustrate the reversed micelle systems. The twin-tailed structural formula of the anionic surfactant bis (2-ethylhexyl) sodium sulfosuccinate, commonly known as AOT, is shown in FIG. 2(a). FIG. 2(b) illustrates the micellar environment with the enzyme encapsulated in the water pools. The head of the arrow indicating the monomer represents the polar hydroxyl group of the phenol. In the micellar environment, the phenol molecules partition to the water-oil interface. Indeed, Fourier Transform Infrared (FTIR) spectra shows a low frequency shift in the AOT carbonyl stretch as a result of hydrogen bonding with phenol hydroxyls. Dynamic light scattering results also indicate a decrease in micelle curvature through such rigidifaction of the interface. The overall schematic of the reaction system is shown in FIG. 3; note that the formed polymer microspheres and their contents ultimately precipitate out from solution.
Thus, one advantage of the present invention is the ability of our approach to polymerization is to align the monomers at the water-oil interface prior to polymerization. In such an alignment, the hydroxyl groups are on the same side of the polymer backbone, as shown in FIG. 1. The proximity of hydroxyl groups presents an opportunity indicating an ease in functionalizing the polymer, i.e., in metal chelation for the preparation of catalytic polymers, or for protein attachment.
We have found that polymerization in reverse micelles results in monomer conversions up to 80% in less than 5 minutes. The polymer precipitate is in the form of spherical particles, both distinct, and interconnected. FIG. 4 is a scanning electron micrograph (SEM) showing such particles.
Spherical morphologies are generated only at specific phase compositions in the micellar system. The situation is shown in FIG. 5, circles indicate the compositions suitable for the formation of polymer microspheres. The data indicates that an AOT/p-ethylphenol ratio of 3/1 can be used as a conservative criterion to reproducibly obtain spherical particles.
When polymerization is conducted in monophasic organic solvents microspheres are not formed spherical morphologies. The micellar environment appears to act as a template during polymerization to shape the microspheres.
Alternatively the spherical morphology may be the consequence of surface energy minimization when there are no internal stress anisotropies in the particle. However, two reasons indicate that templating is indeed prevalent. First, the transition from spherical to nonspherical morphologies in FIG. 5 indicates that adequate surfactant must be present to obtain the microspheres. At the transition point 11 (FIG. 5), we see spherical particles interspersed with nonspherical particles, and when the monomer concentration is further increased, there is no evidence of spheres.
Second, it is possible to destroy morphology by dissolving the polymer in a polar solvent (e.g., dimethyl formamide (DMF) or tetrahydrofuran (THF)) and then recreate the spheres by precipitation in an AOT micellar solution. This can be done both with polymer produced in reversed micelles and polymer produced in monophasic organic solvents. Precipitation in a surfactant-free nonpolar solvent (e.g., isooctane) does not recover the spherical morphology. This suggests surfactant-induced folding of polymer chains, perhaps; as a consequence of hydrogen bonding interactions between polymer hydroxyl groups and the surfactant carbonyl and sulfonate groups. The polymer microsphere morphology is retained as adsorbed surfactant is washed off the polymer particles.
In the following examples, we have used the phase composition AOT 0.5 M and p-ethylphenol 0.15 M for reproducible spherical morphologies, with a w0 (water to AOT molar ratio) of 15. The enzyme level in synthesis was set at 1 mg/ml solution. Usually H2 O2 additions are set at 30% excess of the stoichiometric requirement (0.2 M added in 3 aliquots, to minimize enzyme deactivation).
The size of the polymer particles, 200-800 nm on average, is much larger than the reversed micelle diameter, which varies from 5-15 nm, depending on the water content. This indicates that the particles are not just the consequence of growth around the periphery of the micelle.
Monomer conversion follows the reaction stoichiometry (FIG. 1) quite closely. By controlling the amount of H2 O2 added, it is possible to monitor morphology development. FIG. 6 is a transmission electron micrograph of an early stage. Sufficient H2 O2 was added to complete 25% conversion of a 0.15 M p-ethylphenol monomer and the precipitate was collected after 5 minutes. The development of overall sphericicity can be seen. The interconnected spheres are not of uniform density but made up of dark patched, which are approximately of the size of the reversed micelle. Realizing that the micellar system is dynamic with exchange of material between micelles as a result of micellar collisions, it is believed that the resulting polymer microsphere particles are made up of clusters of smaller particles that may have their origin through growth on the micelle periphery.
The enzyme is trapped in the polymer microspheres and removed from the reversed micelles. The peroxidase is trapped in the polymer as it precipitates, as schematically shown in FIG. 3. In this example a small amount of gold-labeled peroxidase was added to the reversed micellar reaction mixture. The electron-dense gold particles are seen as the dark spots in the transmission electron micrograph of the polymer microspheres (FIG. 7).
In a separate example, a marker enzyme (e.g., cytochrome c) cosolubilized in the micelles is also essentially completely removed from the micelles and trapped in the polymer matrix, as shown by the loss of absorbance of the marker in the supernatant after reaction (FIG. 15).
The enzyme trapped in the polymer microspheres stays catalytically active. In another example, precipitated polymer microspheres with trapped peroxidase were recovered and contacted with a fresh reaction mixture (reversed micellar solution with monomer but without enzyme). Rapid polarization occurred, up to 80% of the initial rate catalyzed by naked peroxidase in reversed micelles. This indicates that the polymer encapsulated enzyme is capable of further catalysis.
The internal density of the polymer microspheres can be controlled by reagent concentration and the duration of reaction. The catalytic activity of trapped peroxidase leads to an interesting observation. Within 5-30 minutes of reaction initiated by H2 O2 added to at least the 100% stoichiometric requirement, the measured monomer conversion is greater than 80%, and a significant polymer precipitate is recovered. Electron micrography of the produced polymer reveals that the overall morphology has already developed at these conversion levels. However, at these short reaction times, direct TEM imaging revealed that the particles resemble spherical shells with openings and diffuse interiors. FIG. 8(a) illustrates this morphology. Both a cluster of interconnected particles at magnification (×75,000) and a single particle at a higher magnification (×133,000) are shown in FIG. 8(a). With longer reaction times there was a dramatic increase in internal density. After about 4-5 hours reaction time, about 50% of the particles appeared dense to the electron beam. After 24 hours the interior reaction time of all particles was completely dense, as shown in FIG. 8(b) (magnification ×133,000).
The increased density is clearly the result of continued polymerization catalyzed by the entrapped enzyme. This probably occurs due to residual monomer, which we assume, can much more easily access the active site than the relatively immobile polymer chains. Polymer molecules may become activated through radical transfer from the monomer to the more resonance stabilized chains, the chains may cross link or extend as a result of further monomer attachment.
The densification can be stopped simply by removing the polymer precipitate after 5-15 minutes reaction and thoroughly washing the precipitate with isoctane and water to remove residual monomer and H2 O2. A better method of controlling densification is to simply reduce H2 O2 addition to 80-90% of stoichiometric requirement for complete monomer conversion. Thus, the internal density of the polymer particles can be controlled.
It has been found that ultrasonication of the diffuse polymer particles obtained at short reaction times results in breakage of connection points and/or eruption of internal material out of the particle, leaving hollow shells behind. The scanning electron micrographs of FIGS. 9(a) and 9(b) illustrate such polymer microspheres with apparently hollow interiors obtained as a result of mild sonication.
Reversed micelles have been proposed as a microenvironment within which to synthesize inorganic material that do not grow beyond the nanometer size range. Enhanced band-gap semiconductor materials (CdS, TiO2), magnetic particles, and the like are examples of novel nanoparticles that have been synthesized in reversed micelles; their synthesis and properties are known in the art.
We made polymer-nanoparticle composites by first synthesizing the nanoparticles in the reversed micelles, then adding monomer, enzyme, and H2 O2 to initiate polymer synthesis. When the polymer microspheres precipitate out they incorporate a significant amount of the nanoparticles.
In one example, particles of superparamagnetic iron oxide have been synthesized in reversed micelles. In such synthesis, the particle size approaches magnetic domain size, leading to thermally induced randomization of magnetic dipole orientations. The particles, therefore, do not exhibit magnetic characteristics in the absence of a field. The term superparamagnetism is usually applied to particles which are nonmagnetic in the absence of a field as paramagnetic materials are, but upon application of an external field exhibit magnetization far in excess of paramagnetic materials.
We have then incorporated these particles into the polymer as described above, allowing polymerization to continue to yield dense microspheres. FIG. 10 is a transmission electron micrograph of a cross-section of a polymer particle. The dark specks are the ferrite particles uniformly distributed across the section. The cross-section also indicates a significant amount of ferrite entrapment (up to 6% of the polymer weight). The particle size increases upon incorporation of the nanoparticles. The particle deformation in the micrograph is an artifact of the sectioning procedure.
The composite polymer iron oxide material had superparamagnetic properties. Magnetization loops for the material, as generated by a superconducting quantum interference device (SQUID) are illustrated in FIGS. 11 (a) and 11 (b). The magnetization vs. field data at 100 degrees Kelvin illustrates that the data is perfectly superimposable as the field is cycled between ±50 kG, with the coercivity Hc =0. This lack of hysteresis is characteristic of superparamagnetic particles or some singledomain particles of very small dimensions. The particles have very small volumes, and thermal fluctuations are sufficient to overcome the anisotropy energy barrier, allowing the magnetization to spontaneously reverse direction. As the temperature is significantly lowered to 4.5 degrees K, the sample starts to display some hysteresis with coercivity Hc about equal to 700 G (FIG. 11 (b)), as the thermal energy is insufficient to overcome the anisotropy energy barrier.
Iron oxide (ferrite) encapsulated in the polymer matrix is suitable for use in magnetic coatings and information storage, color imaging, magnetic chromatography and separations, magnetic refrigeration and ferrofluids, and magnetic resonance imaging. Iron oxide encapsulated in the polymer matrix with specific antibodies is suitable for magnetic isolation, separation and purification of specific antigen containing cells and other biological materials.
The method of preparing polymer-iron oxide composites can be generalized to other oxides. Indium oxide can be incorporated into the polymer matrix to make conductive coatings that are relatively flexible and do not easily flake off as direct metal oxide coatings do. Conductive coatings have applications as charge dissipation systems in minimizing electrostatic charge build up.
Semiconductor nanoparticles may also be incorporated into the polymer matrix.
Soluble polymer has been added to reversed micelles. The presynthesized soluble polymer added to the reversed micelle emulsions encapsulated the intramicellar solutes and precipitated as polymer microspheres containing those solutes.
A polymer was prepared by polymerization of p-ethylphonol in a water in oil reversed micelle system (0.5M AOT in isooctane, w0 =15). A typical reaction mixture composition was 0.15 M p-ethylphenol, an equimolar amount of hydrogen peroxide and 0.5 mg/ml of enzyme (HRP). The reaction was carried out for 10-15 minutes. The reaction mixture was centrifuged, the precipitated collected and washed with isooctane. The precipitated polymer particles were resuspended in isooctane and subjected to mild sonication for 5-10 minutes. The sample was then removed from the sonication bath, dried in the vacuum oven and stored for future use.
Tetracycline was dissolved in water to make a concentrated solution (0.01 g/m1). A small amount (2 ml) of this concentration solution was then added to the dried carrier polymer powder (0.1 g), such that the polymer was completely wetted with the solution.
The thick slurry of the polymer suspension was then agitated for some time to allow proper mixing and finally dried in vacuum oven.
The dried polymer with the trapped tetracycline was accurately weighed and redispersed in a measured volume of water. The suspension was kept agitated by stirring (magnetically). Aliquot of the aqueous suspension were collected over regular intervals of time and assayed for tetracycline content (UV absorbance). Prior to measurement, however, the solution was centrifuged and the supernatant filtered.
Enzymes encapsulated in polymer-microspheres have been shown to be catalytically active and stable. Enzymes were first co-solubilized in reversed micelles with horseradish peroxidase, following which, poly (p-ethylphenol) was enzymatically synthesized. A specific example is the cosolubilization of enzymes which break down organophosphorus compounds. Organophosphorus acids (OPA) and their salts are toxic compounds. Organofluorophosphonates, for example, are extremely toxic and constitute chemical warfare nerve agents such as soman and sarin. Organophosphonates, organophosphinates, and organophosphates include other compounds of this class, which include insectides and pesticides such as parathion, paraoxon, and malathion are considerably less toxic and are often used as surrogates in evaluating decontamination technologies.
Organophosphorus acid hydrolases or phosphotriesterases are able to function in vitro without any cell-mediated cofactors.
We have studied commercially available phosphodiesterase from Crotalus ctrox (Sigma Chemical Company, St. Louis, Mo.) (FIG. 15). The enzyme, when encapsulated in the polymer microspheres showed activity, releasing nitrophenol (measured by the increase in absorbance at 405 nm) from bis (para-nitrophenyl) phosphate with a rough initial rate of 10-12 μmoles nitrophenol produced/mg enzyme/min.
The present invention has been applied to luminescent aromatic monomers to produce highly luminescent polymers. Aromatic compounds, in particular fused ring species, exhibit fluorescence. In addition, polymers and copolymers from multiring or fused ring phenols (e.g.naphthols and hydroxypyrenes) are also electroluminescent. Pyrene, for example, has been studied extensively as a probe of molecular environment, since it has a long singlet lifetime, is able to act as an energy acceptor, and its emission characteristics are sensitive to the chemical environments. But our objective is to enzymatically prepare polymers out of these fluorescent aromatics and attempt to control luminescence over a broad spectral range. The attachment of semiconductor nanoparticles (e.g. CdS) to these polymers expands the applications of these polymers. Fluorescent polymers have applications in optical recording and photolithography, luminescent solar concentrators, laser materials, and even paints and varnishes.
Polymers have been made with 2-naphthol and 1-hydroxypyrene in reversed micelles (the monomer structures are shown in FIG. 12). The polymers are photophysically active and the fluorescence spectra shown in FIG. 14 indicates a significant Stokes shift ranging from about 40 nm (peak at about 450 nm for (poly) 2-naphthol) to about 100 nm (peak at 510 nm for (poly) 1-hydroxypyrene). The copolymer's emission peak lies at an intermediate frequency, indicating that copolymer composition can be used to manipulate emission frequencies.
Enhanced band-gap semiconductor nanoparticles are also suitable for polymer encapsulation. Thiol groups bind irreversibly to nanoparticle CdS and CdSe and are often used to cap surface emissions. First CdS is nucleated in the micelles using well established and simple procedures (bubbling H2 S through CdCl2 containing micelles or mixing CdCl2 micelles with Na2 S micelles). Then hydroxythiophenol (FIG. 12) is used as the monomer for enzymatic polymerization. The monomer binds to CdS and the polymer formed carries the CdS or CdSe with it. Thus this method provides not just an encapsulation of the nanoparticles but a composite material with the CdS or CdSe covalently linked to the polymer. The attachment of CdS or CdSe to the polymer enhanced the red-shift in fluorescence emission. Nanoparticle CdSe for example, emits at a wavelength between 580-620 nm (excitation at 514 nm) depending on particle size.
The present invention is suitable for chromatographic packing material because of account the morphology and particle structure of the polymer. Phenolic polymers may be very effective in binding to hard-to-separate substituted amines through hydrogen bonding of the acidic hydroxyls to amine functionalities.
|Cited Patent||Filing date||Publication date||Applicant||Title|
|US4224179 *||Aug 4, 1978||Sep 23, 1980||Battelle Memorial Institute||Process for the preparation of liposomes in aqueous solution|
|US5324436 *||Mar 12, 1993||Jun 28, 1994||The Administrators Of The Tulane Educational Fund||Use of hydrate formation to control membrane mimetic systems|
|1||Akkara, J.A., Ayyagari, M., Bruno, F., Samuelson, L., John, V.T., Karayigglu, C., Tripathy, S., Marx, K., Rao, D.V.G.L.N., Kaplan, D.L., "Biomimetic Membrane and Interface Templates for Enzyme-Based Polymerization Reactions", Biomimetrics, 2, 331 (1994).|
|2||*||Akkara, J.A., Ayyagari, M., Bruno, F., Samuelson, L., John, V.T., Karayigitoglu, C., Tripathy, S., Marx, K., Rao, D.V.G.L.N., Kaplan, D.L., Biomimetic Membrane and Interface Templates for Enzyme Based Polymerization Reactions , Biomimetrics, 2, 331 (1994).|
|3||Akkara, J.A., Kaplan, D.L., Tripathy, S.K., John, V.T., "Enzyme-Catalyzed Polymerization", in The Polymeric Materials Encyclopedia: Synthesis, Properties and Applications, Salamone, J. Ed., vol. 3, CRC Press, (1996) pp. 2115-2125.|
|4||*||Akkara, J.A., Kaplan, D.L., Tripathy, S.K., John, V.T., Enzyme Catalyzed Polymerization , in The Polymeric Materials Encyclopedia: Synthesis, Properties and Applications, Salamone, J. Ed., vol. 3, CRC Press, (1996) pp. 2115 2125.|
|5||Rao, M., Gonzalez, R.D., John, V.T., Kaplan, D., Akkara, J., "Catalytic and Interfacial Aspects of Enzymatic Polymer Synthesis in Reversed Micelles", Biotechnol. Bioeng., 41, 531 (1993).|
|6||*||Rao, M., Gonzalez, R.D., John, V.T., Kaplan, D., Akkara, J., Catalytic and Interfacial Aspects of Enzymatic Polymer Synthesis in Reversed Micelles , Biotechnol. Bioeng., 41, 531 (1993).|
|Citing Patent||Filing date||Publication date||Applicant||Title|
|US6566469 *||Jun 22, 2000||May 20, 2003||Trustees Of Tufts College||Enzyme-mediated polymerization methods and products|
|US6773823 *||Apr 9, 2001||Aug 10, 2004||University Of New Orleans Research And Technology Foundation, Inc.||Sequential synthesis of core-shell nanoparticles using reverse micelles|
|US6906339||Sep 4, 2002||Jun 14, 2005||Rensselaer Polytechnic Institute||Passivated nanoparticles, method of fabrication thereof, and devices incorporating nanoparticles|
|US6979466 *||Mar 17, 2003||Dec 27, 2005||Leibniz-Institut Fuer Neue Materialien Gemeinnuetzige Gmbh||Nanoscale particles having an iron oxide-containing core enveloped by at least two shells|
|US7253119||May 9, 2005||Aug 7, 2007||Rensselaer Polytechnic Institute||Passivated nanoparticles, method of fabrication thereof, and devices incorporating nanoparticles|
|US7321022||Mar 10, 2006||Jan 22, 2008||Louisiana Tech University Foundation, Inc.||Method for controlling stability of nanofabricated polypeptide multilayer films, coatings, and microcapsules|
|US7348399||Aug 29, 2003||Mar 25, 2008||Louisiana Tech University Foundation, Inc.||Nanofabricated polypeptide multilayer films, coatings, and microcapsules|
|US7411038||May 12, 2006||Aug 12, 2008||Louisiana Tech University Foundation||Artificial red blood cells|
|US7534860||Nov 16, 2007||May 19, 2009||Louisiana Tech University Foundation||Nanofabricated polypeptide multilayer films, coatings, and microcapsules|
|US7538184||Nov 16, 2007||May 26, 2009||Louisiana Tech University Foundation||Method for controlling stability of nanofabricated polypeptide multilayer films, coatings, and microcapsules|
|US7544770||Oct 25, 2006||Jun 9, 2009||Louisiana Tech Foundation, Inc.||Multilayer films, coatings, and microcapsules comprising polypeptides|
|US7550557||Oct 25, 2006||Jun 23, 2009||Louisiana Tech University Foundation, Inc.||Multilayer films, coatings, and microcapsules comprising polypeptides|
|US7834468||Jun 27, 2007||Nov 16, 2010||Rensselaer Polytechnic Institute||Passivated nanoparticles, method of fabrication thereof, and devices incorporating nanoparticles|
|US7892586||Mar 21, 2007||Feb 22, 2011||Suzanne Jaffe Stillman||Water containing soluble fiber|
|US7893198||Apr 9, 2009||Feb 22, 2011||Louisiana Tech University Foundation, Inc.||Multilayer films, coatings, and microcapsules comprising polypeptides|
|US8093038||Sep 17, 2007||Jan 10, 2012||Illinois Institute Of Technology||Apparatus and method for encapsulating pancreatic cells|
|US8178150||Jul 2, 2007||May 15, 2012||Suzanne Jaffe Stillman||Water containing soluble fiber|
|US8257762||Jan 7, 2011||Sep 4, 2012||Suzanne Jaffe Stillman||Water containing soluble fiber|
|US8263327 *||Apr 18, 2008||Sep 11, 2012||Basf Se||Enzymatic method for the production of microcapsules|
|US8360321||Apr 2, 2008||Jan 29, 2013||Fio Corporation||System and method of deconvolving multiplexed fluorescence spectral signals generated by quantum dot optical coding technology|
|US8551763||Oct 10, 2008||Oct 8, 2013||Fio Corporation||Flow focusing method and system for forming concentrated volumes of microbeads, and microbeads formed further thereto|
|US8551786||Jul 9, 2008||Oct 8, 2013||Fio Corporation||Systems and methods for enhancing fluorescent detection of target molecules in a test sample|
|US8597729||Jun 23, 2008||Dec 3, 2013||Fio Corporation||Systems and methods for manufacturing quantum dot-doped polymer microbeads|
|US8663721||May 7, 2012||Mar 4, 2014||Suzanne Jaffe Stillman||Water containing soluble fiber|
|US8771699||Jan 9, 2009||Jul 8, 2014||Magforce Ag||Magnetic transducers|
|US8791053||Sep 26, 2003||Jul 29, 2014||Mpm-Holding Aps||Spatially encoded polymer matrix|
|US9267951||Sep 28, 2010||Feb 23, 2016||King's College London||Micellar compositions for use in biological applications|
|US9302090||Jul 25, 2012||Apr 5, 2016||Applied Medical Technology, Inc.||Photoluminescent coupling|
|US9360476||Dec 19, 2007||Jun 7, 2016||Fio Corporation||Microfluidic system and method to test for target molecules in a biological sample|
|US9408912||Aug 7, 2012||Aug 9, 2016||Magforce Ag||Agglomerating magnetic alkoxysilane-coated nanoparticles|
|US9459200||Aug 31, 2009||Oct 4, 2016||Fio Corporation||Single-use handheld diagnostic test device, and an associated system and method for testing biological and environmental test samples|
|US20020068187 *||Apr 9, 2001||Jun 6, 2002||O'connor Charles J.||Sequential synthesis of core-shell nanoparticles using reverse micelles|
|US20030064104 *||Sep 16, 2002||Apr 3, 2003||Stillman Suzanne Jaffe||Fiber-water with added value delivery systems/elements/additives, addressing specific dietary use(s), and/or medical use(s) for humans and animals|
|US20050069950 *||Aug 29, 2003||Mar 31, 2005||Haynie Donald T.||Method for designing polypeptides for the nanofabrication of thin films, coatings, and microcapsules by electrostatic layer-by-layer self assembly|
|US20050220714 *||Mar 21, 2005||Oct 6, 2005||Susan Kauzlarich||Agents for use in magnetic resonance and optical imaging|
|US20060099247 *||Nov 10, 2004||May 11, 2006||Byrd-Walsh, Llc.||Liquid, gas and/or vapor phase delivery systems|
|US20060127369 *||Sep 26, 2003||Jun 15, 2006||Carlsberg A/S||Spatially encoded polymer matrix|
|US20060147543 *||Mar 10, 2006||Jul 6, 2006||Haynie Donald T||Method for controlling stability of nanofabricated polypeptide multilayer films, coatings, and microcapsules|
|US20060155482 *||Mar 10, 2006||Jul 13, 2006||Haynie Donald T||Method for design of polypeptides for nanofabrication of multilayer films, coatings, and microcapsules|
|US20060205005 *||May 12, 2006||Sep 14, 2006||Haynie Donald T||Artificial red blood cells|
|US20070020771 *||Jun 26, 2006||Jan 25, 2007||Applied Nanoworks, Inc.||Nanoparticles and method of making thereof|
|US20070056465 *||Mar 3, 2004||Mar 15, 2007||Rensselaer Polytechnic Institute||Rapid generation of nanoparticles from bulk solids at room temperature|
|US20070077275 *||Oct 25, 2006||Apr 5, 2007||Haynie Donald T||Multilayer films, coatings, and microcapsules comprising polypeptides|
|US20070077276 *||Oct 25, 2006||Apr 5, 2007||Haynie Donald T||Multilayer films, coatings, and microcapsules comprising polypeptides|
|US20070224490 *||Oct 19, 2004||Sep 27, 2007||Janssen Catharina P||Suspension for the Generation of a Current of Electrons and the Use and the Preparation Thereof|
|US20070248826 *||Jun 27, 2007||Oct 25, 2007||Rensselaer Polytechnic Institute||Passivated nanoparticles, method of fabrication thereof, and devices incorporating nanoparticles|
|US20070258907 *||Apr 24, 2007||Nov 8, 2007||Davis Mark E||Polymer-coated paramagnetic particles|
|US20080245769 *||Jul 17, 2007||Oct 9, 2008||Applied Nanoworks, Inc.||Nanoparticles and method of making thereof|
|US20090075359 *||Sep 17, 2007||Mar 19, 2009||Hatziavramidis Dimitri T||Apparatus and method for encapsulating pancreatic cells|
|US20090233074 *||Apr 9, 2009||Sep 17, 2009||Louisiana Tech University Research Foundation, a division of the Louisiana Tech University||Multilayer Films, Coatings, and Microcapsules Comprising Polypeptides|
|US20100021937 *||Feb 13, 2007||Jan 28, 2010||Fio Corporation||Method for detecting pathogens using microbeads conjugated to biorecognition molecules|
|US20100120617 *||Apr 18, 2008||May 13, 2010||Basf Se||Enzymatic Method for the Production of Microcapsules|
|US20100151443 *||Dec 19, 2007||Jun 17, 2010||Fio Corporation||Microfluid system and method to test for target molecules in a biological sample|
|US20100257027 *||Jul 23, 2008||Oct 7, 2010||Fio Corporation||Method and system for collating, storing, analyzing and enabling access to collected and analyzed data associated with biological and environmental test subjects|
|US20110052609 *||Jan 9, 2009||Mar 3, 2011||Norbert Waldoefner||Magnetic transducers|
|US20110053278 *||Jul 9, 2008||Mar 3, 2011||Fio Corporation||Systems and methods for enhancing fluorescent detection of target molecules in a test sample|
|US20110081643 *||Oct 10, 2008||Apr 7, 2011||Sebastian Fournier-Bidoz||Flow Focusing Method and System for Forming Concentrated Volumes of Microbeads, and Microbeads Formed Further Thereto|
|US20110165688 *||Aug 31, 2009||Jul 7, 2011||Fio Corporation||Single-use handheld diagnostic test device, and an biological and environmental test samples|
|US20110230343 *||Oct 14, 2009||Sep 22, 2011||Basf Se||Method for the Manufacture of Microparticles Comprising an Effect Substance|
|CN100401572C||Oct 19, 2004||Jul 9, 2008||卡特琳娜·菲律宾亚·詹森||Suspension for the generation of a current of electrons and the use and the preparation thereof|
|DE10256085A1 *||Nov 29, 2002||Jun 17, 2004||Henkel Kgaa||Magnetische Mikrobizide|
|DE102014006519A1||May 3, 2014||Nov 5, 2015||Smart Material Printing B.V.||Verwendung magnetischer und/oder magnetisierbarer, polymerer Mikro- und/oder Nanocomposite zur Herstellung komplexer, magnetischer und/oder magnetisierbarer Formteile mithilfe additiver Fabrikatoren|
|WO2005038968A2 *||Oct 19, 2004||Apr 28, 2005||Catharina Philippina Janssen||Suspension for the generation of a current of electrons and the use and the preparation thereof|
|WO2005038968A3 *||Oct 19, 2004||Jun 2, 2005||Catharina Philippina Janssen||Suspension for the generation of a current of electrons and the use and the preparation thereof|
|WO2006002641A1 *||Jul 1, 2005||Jan 12, 2006||Versamatrix A/S||Spherical radiofrequency-encoded beads|
|WO2008132067A2 *||Apr 18, 2008||Nov 6, 2008||Basf Se||Enzymatic method for the production of microcapsules|
|WO2008132067A3 *||Apr 18, 2008||Dec 31, 2008||Basf Se||Enzymatic method for the production of microcapsules|
|WO2011039535A3 *||Sep 28, 2010||May 26, 2011||King's College London||Micellar compositions for use in biological applications|
|U.S. Classification||264/4.1, 264/4.6, 427/213.36, 264/4.7, 427/213.3, 264/4.33, 264/4.3, 427/213.34, 427/213.31, 427/215|
|International Classification||B01J13/18, A61K9/51|
|Feb 24, 1997||AS||Assignment|
Owner name: ARMY, UNITED STATES OF AMERICA, THE, AS REPRESENTE
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:AKKARA, JOSEPH A.;KAPLAN, DAVID L.;REEL/FRAME:008368/0838;SIGNING DATES FROM 19960924 TO 19961002
|Apr 9, 2003||FPAY||Fee payment|
Year of fee payment: 4
|Jun 27, 2007||FPAY||Fee payment|
Year of fee payment: 8
|Sep 12, 2011||REMI||Maintenance fee reminder mailed|
|Feb 8, 2012||LAPS||Lapse for failure to pay maintenance fees|
|Mar 27, 2012||FP||Expired due to failure to pay maintenance fee|
Effective date: 20120208